Week 15&16 Protein Purification Objective: The purpose of this lab is to purify the protein using the affinity tag and Ni-NTA resin.
Protein Expression
Objective: The purpose of this lab is to make proteins for enzyme assays.
Figure 1. OD600 of bacterial cultures measured using Vernier Visible Spectrum (Chipper).
Analysis: After growing the bacterial culture overnight, the OD600 of the culture was measured using the Vernier Visible Spectrum (Chipper). At 600nm, the OD600 reached 0.496. IPTG was added and the proceed to the induction step. The pellet was harvested by centrifugation and weight approximately 2.44g. After sanitation, the sample was spun down to remove the soluble fraction for purification.
Week 13&14 Protein Expression
Cloning - Cohesive End Generation, Annealing & Transformation Objective: The purpose of this procedure is to prepare PCR inserts and accepting vector for cloning.
Figure 1. Plate A of H. pylori of alpha carbonic anhydrase with pNIC-Bsa4 on an LB+KAN+SUC plate from Tube A samples: 2 microliters of PCR insert and 4 microliters of accepting vector.
Figure 2. Plate A of H. pylori of alpha carbonic anhydrase with pNIC-Bsa4
on an LB+KAN+SUC plate from Tube A samples: 1 microliters of PCR insert
and 7 microliters of accepting vector.
Analysis: The cohesive end generation on PCR inserts and on the accepting vector were done in tandem in order to prepare the samples for the next step - annealing and transformation. Two plates from made each from Tube A and Tube B, containing the different amounts of PCR inserts and the accepting vector. Both plates showed no colonies. The next step will be to move on to the surrogate and start protein expression.
Great work. Did you submit to sequencing? -UM
Week 11&12 Cloning - Preparation of pNIC-Bsa4 as Accepting Vector
Objective: The purpose of this lab is to prepare the plasmid as an accepting vector by cutting with the restriction enzyme BsaI-HF in order to proceed to cohesive end generation.
Figure 1. Concentration of DNA plasmid as the accepting vector. Trial 1.
Figure 2. Concentration of DNA plasmid as the accepting vector. Trial 2.
Analysis: The concentrations for pNIC-Bsa4 as the accepting vector were 66.3 ng/uL and 68.0 ng/uL, respectively. The next step would be to perform cohesive end generation with the plasmid and PCR product.
Plasmid DNA Purification by Midi Prep Protocol
Objective: The purpose of this protocol is to extract the DNA from transformed bacterial cells.
Figure 1. Concentration of the final eluted DNA after Midiprep.
Analysis: After the plasmid DNA was extracted and purified using Midi prep, the concentration was taken and came out to be 115.5 ng/uL. The 260/280 was 1.90, and the 260/230 was 2.79. The next step will be to prepare for cloning by preparing the pNIC-Bsa4 as accepting vector.
Transformation of Competent Cells for Plasmid Prep of pNIC-Bsa4
Objective: The purpose of this lab is to transform the bacterial cells with plasmid DNA to make more plasmid DNA.
Figure 1: Pellets containing the DNA after incubation.
Analysis: Bacterial colonies were put in LB media, placed in the 37 degrees C shaking incubator overnight, and centrifuged the next day in order to retain the pellet. Next step was to purify the plasmid DNA by Midi prep.
Making More PCR Product
Objective: Made more PCR Cleanup for second trial of cloning.
Primary PCR -> Secondary PCR -> PCR^2 -> PCR Cleanup
Figure 1. Concentration of PCR Cleanup.
Figure 2. Concentration of PCR Cleanup.
Analysis: Because the first trial of cloning failed, more PCR product was needed in order to proceed to the second trial of getting a positive clone. After PCR Cleanup, the average concentration was measured and came out to be 197.3 ng/uL. The 260/280 was 1.83 and 1.81, respectively. The 260/230 was 2.17 and 2.06, respectively. The next step will be to transform the bacteria with plasmid DNA.
DNA Sequencing
Figure 1. DNA sequencing request form.
Analysis: After getting the results back, BLAST all of the results against the original DNA sequence. However, there was one mismatch in the beginning of the sequence, therefore, there was no positive clone.
Very good! Looks like you got a lot done in lab the past two weeks. Hope you get a positive clone. -UM
Week 9 & 10 Miniprep & Nanodrop
Objective: The purpose of Miniprep is to extract and purify the plasmid DNA from the cell. Nanodrop was performed to determine the concentrations of the DNA.
Figure 1. Concentration of the final eluted DNA of sample 3 from Tube A after Miniprep.
Figure 2. Concentration of the final eluted DNA of sample 4 from Tube A after Miniprep.
Figure 3. Concentration of the final eluted DNA of sample 7 from Tube B after Miniprep.
Analysis: All eight tubes contained pellets, but the three pellets (Figures 1-3) showed the higher concentrations. Sample 7 from Tube B had the highest concentration among the eight tubes with a 260/280 of 1.83 and 260/230 of 1.63. Possibly low concentration may be the result of adding 100 microliters of the elution solution instead of 50 microliters.
Master Plate
Objective: The purpose of making the master plate is to conglomerate the PCR inserts and accepting vector to get positive matches from DNA sequencing.
Figure 1. H. pylori of alpha carbonic anhydrase containing the accepting vector on LB+KAN+SUC plate. 1-6: colonies from Plate A. 7-8: colonies from Plate B.
Analysis: 1-8 all had one colony from overnight in the incubator. When all eight tubes were spun down, all of the tubes formed a pellet.
pNIC-Bsa4 Cloning - Cohesive End Generation, Annealing & Transformation
Objective: The purpose of this procedure is to prepare PCR inserts and accepting vector for cloning.
Figure 1. Plate A of H. pylori of alpha carbonic anhydrase with pNIC-Bsa4 on an LB+KAN+SUC plate from Tube A samples: 2 microliters of PCR insert and 4 microliters of accepting vector.
Figure 2. Plate B of H. pylori of alpha carbonic anhydrase with pNIC-Bsa4 on an LB+KAN+SUC plate from from Tube B samples. 2 microliters of PCR inserts and 8 microliters of accepting vector.
Analysis: The cohesive end generation on PCR inserts and on the accepting vector were done in tandem in order to prepare the samples for the next step - annealing and transformation. Two plates from made each from Tube A and Tube B, both containing the same amount of PCR inserts, but different amounts of the accepting vector. Plate A showed more colonies than Plate B.
Virtual Screening - Creating Homology Model
Amino Acid Sequence of Target: MKKTFLIALALTASLIGAENTKWDYKNKENGPHRWDKLHKDFEVCKSGK SQSPINIEHYYHTQDKADLQFKYAASKPKAVFFTHHTLKASFEPTNHIN YRGHDYVLDNVHFHAPMEFLINNKTRPLSAHFVHKDAKGRLLVLAIGFE EGKENPNLDPILESVQKKQNFKEVALDAFLPKSINYYHFNGSLTAPPCT EGVAWFVIEEPLEVSAKQLAEIKKRMKNSPNQRPVQPDYNTVIIKSSAETR
Top Template PDB ID: 4g7aB Identities=39% Positives=55%
pNIC-Bsa4 Cloning - Preparation of pNIC-Bsa4 as Accepting Vector
Objective: The purpose of this experiment was to transfer the gene of interest into the protein expression vector pNIC-Bsa4.
Figure 1. The concentration of the plasmid after PCR cleanup to prepare the DNA as an accepting vector.
Analysis: After Midiprep, pNIC-Bsa4 was prepared to be used as the accepting vector for cloning. The concentration was high enough to proceed to the next step of cloning.
Re-do of Midiprep
Objective: The purpose of this lab was to extract the DNA from transformed bacterial cells in order to obtain just the plasmid.
Trial 4: 10/25/13
Figure 1. Concentration of the eluted DNA after Midiprep.
Analysis: After four trials of growing pNIC-Bsa4 and performing Midiprep, the concentration of the final eluted DNA was high enough to proceed to the next step.
Nice work. Could use a little more analysis. Try the pNIC again, the concentrations looked a little low. -UM
Week 7 & 8 Re-do of Transforming Bacterial Cells
Objective: The purpose of this was to transform the E. coli DN5α with the plasmid pBIC-Bsa4 to make more plasmid DNA
Figure 1: Pellets containing the DNA after incubation.
Analysis: One colony that grew from each plate were placed in LB media and Kanamycin stock was added and was placed in the 37 degree shaker overnight for approximately 16 hour. The next day, in the morning, the liquids were centrifuged and the bacteria pellet was placed in the -20 degree freezer.
pNIC-Bsa4 Cloning - Preparation of pNIC-Bsa4 as Accepting Vector
Objective: The purpose of this experiment was to transfer the gene of interest into the protein expression vector pNIC-Bsa4.
Trial 1: 10/16/13
Figure 1: The concentration of the extracted DNA that was incubated with pNIC-Bsa4
The concentration was 2.8 ng/ul, which is a very low concentration.
Trial 2: 10/17/13
Figure 2: The concentration of the extracted DNA that was incubated with pNIC-Bsa4.
The concentration came out to be 2.1 ng/ul.
Trial 3: 10/18/13
Figure 3: The concentration of the extracted DNA that was incubated with pNIC-Bsa4.
The concentration came out to be 8.3 ng/ul.
Analysis: To determine if pNIC-Bsa4 could be used as an accepting vector for the gene of interest, PCR clean up was performed to see if the concentration was high enough to transfer the gene into the vector. In the first two trials, the incorrect wash solution was used - ethanol was not contained in the bottle - so the concentration was very low. In the third trial, it was assumed that a higher concentration would be obtained by using the correct ethanol-containing wash solution, however, the concentration was low again - 8.3 ng/ul. Thus, the bacteria culture had to be grown again.
MidiPrep
Objective: The purpose of this lab was to extract the DNA from transformed bacterial cells in order to obtain just the plasmid.
Figure 1: The concentration of the final eluted DNA after Midi Prep.
Analysis: By using the Midi Prep kit, the DNA from the bacterial cells were extracted. After using several necessary buffers inside the kit, the cell was lysed, releasing every component except the plasmid, such the cell wall, cell membrane, proteins and other inclusions. The concentration came out to be 23.5 ng/ul, the 260/280 value was 2.02, and the 260/230 value was -1.88. By looking at the purity values, it can be determined that the DNA was contaminated from other contaminants.
PCR Cleanup & Nanodrop
Objective: The purpose of this lab was to determine the concentration of the DNA by using spectrophotometry.
Analysis: The concentration of the final DNA product from using nanodrop came out to be 67.3 ng/ul, which is a pretty high concentration.At wavelength of 230nm, the absorbance was 0.635. The purity was also determined by the 260/280 and 260/230 values, which tells how the pure the DNA is in relation to the protein, and in the presence of other contaminants, respectively. The 260/280 value was 1.80 and 260/230 was 2.12, which tells the the DNA is relatively pure.
Great work! be sure to include a ladder image. Great job on the progress! - Michael T.
Week 5 & 6 Transformation of Competent Cells for Plasmid Prep of pNIC-Bsa4
Objective: The purpose of this was to transform the E. coli DN5α with the plasmid pBIC-Bsa4 to make more plasmid DNA.
Figure 1: Pellets containing bacteria and LB+KAN, two of each with different density.
Analysis: The procedure took three days to perform. On the first day, the bacteria was added to LB media and was incubated overnight. The next day, in the afternoon, one colony that grew from each plate were placed in LB media and Kanamycin stock was added and was placed in the 37 degree shaker overnight for approximately 16 hour. The third day, in the morning, the liquids were centrifuged and the bacteria pellet was placed in the -20 degree freezer.
LB Media and LB plate
Objective: The purpose of this lab was to make LB media and LB plates to perform the transformation step.
Analysis: The LB media was made by adding Bacto-tryptone, Bacto-yeast extract, and NaCl, and autoclaving. The LB plates were made by adding the same compounds but, in addition, including the antibiotic, Kanamycin stock. This lab was performed for the next step - transformation of competent cells for plasmid preparation of pNIC-Bsa4.
PCR Primer Overlap - PCR Squared
Objective: The purpose of this lab was to produce lots of copies of the PCR products because most of them will be lost during the PCR cleanup.
Image 1: 1kb DNA ladder.
Figure 1: PCR Squared reaction on 1% agarose gel.
Lane 1: 1kb DNA ladder
Lanes 2-4: PCR Squared reactions (contain 5X rxn buffer, dNTP, secondary PCR reaction, Forward and Reverse primers, Q5, and nanopure water)
Analysis: The PCR squared reactions which amplified the reaction is shown on the 1% agarose gel. The 100bp DNA ladder was not in the -20 degrees freezer, so instead the 1kb DNA ladder was used. However, the size of the gene came out to be around 741 nt. Many copies were made because a lot of the products will eventually be lost in PCR cleanup.
PCR Primer Overlap - Secondary PCR
Objective: The purpose of this experiment was to insert the forward and reverse primers into the primary PCR reaction to piece them together.
Figure 1: PCR Secondary reaction next to 100 bp DNA ladder shown on 1% agarose gel.
Lane 1: Empty
Lane 2: 100 bp DNA ladder
Lane 3: Secondary PCR reaction.
Analysis: Lane 3, according to the image, showed the secondary PCR reaction with a size of the gene was about 741 nt, which is similar to the size of the gene from the output file.
Week 3 & 4
Jessica - good work - include a ladder image - Dr. B 092713
PCR Primer Overlap - Primary PCR
Objective: The purpose of this experiment was to fill in the gaps between the oligos and piece together a full length DNA for the gene by conducting PCR.
Figure 1: Primary PCR reaction shown on 1% agarose gel.
Image 1: 100 bp DNA ladder.
Lane 1: Empty
Lane 2: 100 bp DNA ladder
Lane 3: Primary PCR reactions on 1% agarose gel.
Analysis: The Lane 3 shows the overlapping of the DNA with the nucleotides filling in the gaps in the oligo mix. The gel needs to run longer so the reaction comes down and appears as a more smeared image.
Ordered Forward and Reverse Primers of Gene:
PCR Primer Overlap - Oligo Mix
Objective: The purpose of this lab was to prepare an oligo mix in order to synthesize the gene of interest in future experiments.
Gene: HP Alpha CA
Analysis: The primers that were ordered were put together to create an oligo mix for future experimentation. In the later steps, PCR will be utilized to fill in the gaps between the oligos and piece together a full length of DNA for the gene.
PCR Primer Design Tails for pNIC-Bsa4 Cloning
Objective: The purpose of this lab was to design a forward and reverse primer from the gene (Alpha Carbonate Dehydratase) sequence obtained from the CDS and to synthesize compatible ends for LIC. After designing the primers, the gene would be inserted into the pNIC-Bsa4 as the accepting vector and cut with enzyme BsaI for further expression
0.4 uM Oligo concentration 50 mM Na+ 0.3 mM dNTPs 0 mM Mg++
Figure 2: Gene of interest (Alpha Carbonate Dehydratase) cut with enzyme BsaI.
Figure 3: Gene inserted in pNIC-Bsa4 and cut with enzyme BsaI.
Analysis: The coding sequence for Alpha Carbonate Dehydratase was used along with both upstream and downstream primers to perform LIC cloning. The sequence was then inserted in the pNIC-Bsa4 and was cut with enzyme BsaI, giving two restriction sites. The forward primer has a GC content of 36.7% with 30bp. The reverse primer had a GC content of 42.4% with 33bp and had a much higher melting point of 70.3 degrees Celcius than that of the forward primer.
Restriction Enzyme Digest
Objective: The purpose of this lab was to digest the pGBR22 plasmid with certain restriction enzymes - EcoRI, PvuII, and both, and to visualize the restriction sites on the agarose gel.
Figure 1: Agarose gel image of pGBR22 plasmid with certain restriction enzymes.
Lane 1: Empty.
Lane 2: 1kb DNA ladder.
Lane 3: Uncut plasmid containing diluted plasmid and nanopure water.
Lane 4: pGBR22 plasmid cut with EcoRI.
Lane 5: pGBR22 plasmid cut with PvuII.
Lane 6: pGBR22 plasmid cut with EcoRI + PvuII.
Analysis: Compared to the 1kb DNA ladder, there seemed to be no plasmid present for the uncut plasmid. Lane 4, plasmid cut with EcoRI, showed the plasmid at 4.0kb with one restriction site. Lane 5, cut with enzyme PvuII, showed the plasmid at 3.0kb and 1.0kb - two restriction enzymes. Lane 6, cut with EcoRI and PvuII, showed the plasmid at three restriction sites - 3.0kb, slightly below 1.0kb and slightly below 0.5kb. The intensity of the UV light appeared strongest where the DNA was most present.
PCR Trial 2
Figure 1: Agarose gel containing 100bp DNA ladder and four different dilutions of DNA primer.
Lane 1:Empty.
Lane 2: 100bp DNA ladder.
Lane 3: Tube A consisting 1:1000 dilution template with 18 uL of nanopure water; 0.3ng of plasmid.
Lane 4: Tube B consisting 1:1000 dilution template with 9 uL of nanopure water; 3ng of plasmid.
Lane 5: Tube C consisting 1:100 dilution template with 9 uL of nanopure water; 30ng of plasmid.
Lane 6: Tube D consisting of no DNA template.
Analysis:
The size of the DNA is around 1000bp. The intensity of the UV light is stronger in lanes B and C because more DNA plasmid is present in those locations. The pGBR22 also consisted of M13 Forward and M13 Reverse primers.
Week 1 & 2
PCR Primer Design for Primer Overlap Assembly PCR
Objective:
The purpose of the experiment was to design a set of oligo primers of both reverse and forward for further experimentation.
Objective:
The purpose of this experiment was to determine the concentration and absorbance of the DNA plasmid.
Figure 1. Trial 1 of pGBR22 using Spectrophotometer with absorbance of 3.358 and concentration of 411.8 ng/uL.
Figure 2. Trial 2 of pGBR22 using Spectrophotometer with absorbance of 3.010 and concentration of 383.3 ng/uL.
Results/Conclusion:
The average concentration of the protein pGBR22 came out to be 397.6 ng/uL, which is pretty close to the originally obtained concentration of 365 ng/uL. Also, the purity of the sample in relation to protein (260/280) came out to an average of 1.88 and the purity in relation to other contaminants (260/230) came out to an average of 2.50. Thus, the protein is relatively pure.
Submit to DNA Sequencing
pGBR22 Forward Primer Sequence: NNNNNNNNNNNNNGGNGANTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGNTTTTAGTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTAGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTNGNAAANCTGTCNTGCCAGCTGCNTNATGAATCGGCNANGCGCGGGGANANGCNGNTNGCGTATTGGGCGCTCNTCNNTTCCTCNNTCANTGACTCNNNNCNCTNGNCNTNNNNNNNNNNNNNGNNTCANCTCNNTNNANGNGNANTANNNNNNNNNNNNNNCNNGNNNANNNNNNANNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAAANNNCNNNNNNNNNNNNNNTTTTNNNNNNNNNNNN
Objective:
The purpose of this lab was to, by using the Forward and Reverse Primers, intensify the coding sequence of the purple protein, and also to test four different samples by diluting the DNA primer.
Figure1 . Agarose gel consisting the gel ladder and four different samples dilutions of DNA primer.
Lane 1: Empty
Lane 2: 100 bp ladder
Lane 3: Tube A consisting 1:1000 dilution template with 18 uL of nanopure water.
Lane 4: Tube B consisting 1:1000 dilution template with 9 uL of nanopure water.
Lane 5: Tube C consisting 1:100 dilution template with 9 uL of nanopure water.
Lane 6: Tube D consisting of no DNA template.
Analysis/Conclusion:
After running the gel, the agarose gel went through the UV imaging box. By comparing to the ladder, the diluted primers seem to be the size of 100bp. However, the DNA may not have been diluted correctly because there are so much DNA present on top.
Analyzing DNA Sequencing
Objective:
The purpose of this lab was to analyze and determine the DNA sequence of the plasmid by using BLAST.
Figure 1. The reverse complement of the protein pGBR22.
Protein Purification
Objective: The purpose of this lab is to purify the protein using the affinity tag and Ni-NTA resin.
Protein Expression
Objective: The purpose of this lab is to make proteins for enzyme assays.
Analysis: After growing the bacterial culture overnight, the OD600 of the culture was measured using the Vernier Visible Spectrum (Chipper). At 600nm, the OD600 reached 0.496. IPTG was added and the proceed to the induction step. The pellet was harvested by centrifugation and weight approximately 2.44g. After sanitation, the sample was spun down to remove the soluble fraction for purification.
Week 13&14
Protein Expression
Cloning - Cohesive End Generation, Annealing & Transformation
Objective: The purpose of this procedure is to prepare PCR inserts and accepting vector for cloning.
Figure 2. Plate A of H. pylori of alpha carbonic anhydrase with pNIC-Bsa4
on an LB+KAN+SUC plate from Tube A samples: 1 microliters of PCR insert
and 7 microliters of accepting vector.
Analysis: The cohesive end generation on PCR inserts and on the accepting vector were done in tandem in order to prepare the samples for the next step - annealing and transformation. Two plates from made each from Tube A and Tube B, containing the different amounts of PCR inserts and the accepting vector. Both plates showed no colonies. The next step will be to move on to the surrogate and start protein expression.
Great work. Did you submit to sequencing? -UM
Week 11&12
Cloning - Preparation of pNIC-Bsa4 as Accepting Vector
Objective: The purpose of this lab is to prepare the plasmid as an accepting vector by cutting with the restriction enzyme BsaI-HF in order to proceed to cohesive end generation.
Analysis: The concentrations for pNIC-Bsa4 as the accepting vector were 66.3 ng/uL and 68.0 ng/uL, respectively. The next step would be to perform cohesive end generation with the plasmid and PCR product.
Plasmid DNA Purification by Midi Prep Protocol
Objective: The purpose of this protocol is to extract the DNA from transformed bacterial cells.
Analysis: After the plasmid DNA was extracted and purified using Midi prep, the concentration was taken and came out to be 115.5 ng/uL. The 260/280 was 1.90, and the 260/230 was 2.79. The next step will be to prepare for cloning by preparing the pNIC-Bsa4 as accepting vector.
Transformation of Competent Cells for Plasmid Prep of pNIC-Bsa4
Objective: The purpose of this lab is to transform the bacterial cells with plasmid DNA to make more plasmid DNA.
Figure 1. Pellets containing E.coli DH5α + pNIC-Bsa4.
Analysis: Bacterial colonies were put in LB media, placed in the 37 degrees C shaking incubator overnight, and centrifuged the next day in order to retain the pellet. Next step was to purify the plasmid DNA by Midi prep.
Making More PCR Product
Objective: Made more PCR Cleanup for second trial of cloning.
Primary PCR -> Secondary PCR -> PCR^2 -> PCR Cleanup
Analysis: Because the first trial of cloning failed, more PCR product was needed in order to proceed to the second trial of getting a positive clone. After PCR Cleanup, the average concentration was measured and came out to be 197.3 ng/uL. The 260/280 was 1.83 and 1.81, respectively. The 260/230 was 2.17 and 2.06, respectively. The next step will be to transform the bacteria with plasmid DNA.
DNA Sequencing
Analysis: After getting the results back, BLAST all of the results against the original DNA sequence. However, there was one mismatch in the beginning of the sequence, therefore, there was no positive clone.
Very good! Looks like you got a lot done in lab the past two weeks. Hope you get a positive clone. -UM
Week 9 & 10
Miniprep & Nanodrop
Objective: The purpose of Miniprep is to extract and purify the plasmid DNA from the cell. Nanodrop was performed to determine the concentrations of the DNA.
Analysis: All eight tubes contained pellets, but the three pellets (Figures 1-3) showed the higher concentrations. Sample 7 from Tube B had the highest concentration among the eight tubes with a 260/280 of 1.83 and 260/230 of 1.63. Possibly low concentration may be the result of adding 100 microliters of the elution solution instead of 50 microliters.
Master Plate
Objective: The purpose of making the master plate is to conglomerate the PCR inserts and accepting vector to get positive matches from DNA sequencing.
Analysis: 1-8 all had one colony from overnight in the incubator. When all eight tubes were spun down, all of the tubes formed a pellet.
pNIC-Bsa4 Cloning - Cohesive End Generation, Annealing & Transformation
Objective: The purpose of this procedure is to prepare PCR inserts and accepting vector for cloning.
Analysis: The cohesive end generation on PCR inserts and on the accepting vector were done in tandem in order to prepare the samples for the next step - annealing and transformation. Two plates from made each from Tube A and Tube B, both containing the same amount of PCR inserts, but different amounts of the accepting vector. Plate A showed more colonies than Plate B.
Virtual Screening - Creating Homology Model
Amino Acid Sequence of Target:
MKKTFLIALALTASLIGAENTKWDYKNKENGPHRWDKLHKDFEVCKSGK
SQSPINIEHYYHTQDKADLQFKYAASKPKAVFFTHHTLKASFEPTNHIN
YRGHDYVLDNVHFHAPMEFLINNKTRPLSAHFVHKDAKGRLLVLAIGFE
EGKENPNLDPILESVQKKQNFKEVALDAFLPKSINYYHFNGSLTAPPCT
EGVAWFVIEEPLEVSAKQLAEIKKRMKNSPNQRPVQPDYNTVIIKSSAETR
Top Template PDB ID: 4g7aB
Identities=39%
Positives=55%
Template Molprobity Scores
Clashscore=7.73
Molprobity score=1.69
Homology Molprobity Scores
Clashscore=7.16
Molprobity score=1.66
pNIC-Bsa4 Cloning - Preparation of pNIC-Bsa4 as Accepting Vector
Objective: The purpose of this experiment was to transfer the gene of interest into the protein expression vector pNIC-Bsa4.
Figure 1. The concentration of the plasmid after PCR cleanup to prepare the DNA as an accepting vector.
Analysis: After Midiprep, pNIC-Bsa4 was prepared to be used as the accepting vector for cloning. The concentration was high enough to proceed to the next step of cloning.
Re-do of Midiprep
Objective: The purpose of this lab was to extract the DNA from transformed bacterial cells in order to obtain just the plasmid.
Trial 4: 10/25/13
Figure 1. Concentration of the eluted DNA after Midiprep.
Analysis: After four trials of growing pNIC-Bsa4 and performing Midiprep, the concentration of the final eluted DNA was high enough to proceed to the next step.
Nice work. Could use a little more analysis. Try the pNIC again, the concentrations looked a little low. -UM
Week 7 & 8
Re-do of Transforming Bacterial Cells
Objective: The purpose of this was to transform the E. coli DN5α with the plasmid pBIC-Bsa4 to make more plasmid DNA
Tube 1-4: pellet containing E.coli DH5α + pNIC-Bsa4 and LB+KAN - 10 microliters
Analysis: One colony that grew from each plate were placed in LB media and Kanamycin stock was added and was placed in the 37 degree shaker overnight for approximately 16 hour. The next day, in the morning, the liquids were centrifuged and the bacteria pellet was placed in the -20 degree freezer.
pNIC-Bsa4 Cloning - Preparation of pNIC-Bsa4 as Accepting Vector
Objective: The purpose of this experiment was to transfer the gene of interest into the protein expression vector pNIC-Bsa4.
Trial 1: 10/16/13
The concentration was 2.8 ng/ul, which is a very low concentration.
Trial 2: 10/17/13
The concentration came out to be 2.1 ng/ul.
Trial 3: 10/18/13
The concentration came out to be 8.3 ng/ul.
Analysis: To determine if pNIC-Bsa4 could be used as an accepting vector for the gene of interest, PCR clean up was performed to see if the concentration was high enough to transfer the gene into the vector. In the first two trials, the incorrect wash solution was used - ethanol was not contained in the bottle - so the concentration was very low. In the third trial, it was assumed that a higher concentration would be obtained by using the correct ethanol-containing wash solution, however, the concentration was low again - 8.3 ng/ul. Thus, the bacteria culture had to be grown again.
MidiPrep
Objective: The purpose of this lab was to extract the DNA from transformed bacterial cells in order to obtain just the plasmid.
Analysis: By using the Midi Prep kit, the DNA from the bacterial cells were extracted. After using several necessary buffers inside the kit, the cell was lysed, releasing every component except the plasmid, such the cell wall, cell membrane, proteins and other inclusions. The concentration came out to be 23.5 ng/ul, the 260/280 value was 2.02, and the 260/230 value was -1.88. By looking at the purity values, it can be determined that the DNA was contaminated from other contaminants.
PCR Cleanup & Nanodrop
Objective: The purpose of this lab was to determine the concentration of the DNA by using spectrophotometry.
Analysis: The concentration of the final DNA product from using nanodrop came out to be 67.3 ng/ul, which is a pretty high concentration.At wavelength of 230nm, the absorbance was 0.635. The purity was also determined by the 260/280 and 260/230 values, which tells how the pure the DNA is in relation to the protein, and in the presence of other contaminants, respectively. The 260/280 value was 1.80 and 260/230 was 2.12, which tells the the DNA is relatively pure.
Great work! be sure to include a ladder image. Great job on the progress! - Michael T.
Week 5 & 6
Transformation of Competent Cells for Plasmid Prep of pNIC-Bsa4
Objective: The purpose of this was to transform the E. coli DN5α with the plasmid pBIC-Bsa4 to make more plasmid DNA.
(starting from left)
Tube 1 & 2: pellet containing E.coli DH5α + pNIC-Bsa4 and LB+KAN - 10 microliters
Tube 3 & 4: pellet containing E.coli DH5α + pNIC-Bsa4 and LB+KAN - 50 microliters
Analysis: The procedure took three days to perform. On the first day, the bacteria was added to LB media and was incubated overnight. The next day, in the afternoon, one colony that grew from each plate were placed in LB media and Kanamycin stock was added and was placed in the 37 degree shaker overnight for approximately 16 hour. The third day, in the morning, the liquids were centrifuged and the bacteria pellet was placed in the -20 degree freezer.
LB Media and LB plate
Objective: The purpose of this lab was to make LB media and LB plates to perform the transformation step.
Analysis: The LB media was made by adding Bacto-tryptone, Bacto-yeast extract, and NaCl, and autoclaving. The LB plates were made by adding the same compounds but, in addition, including the antibiotic, Kanamycin stock. This lab was performed for the next step - transformation of competent cells for plasmid preparation of pNIC-Bsa4.
PCR Primer Overlap - PCR Squared
Objective: The purpose of this lab was to produce lots of copies of the PCR products because most of them will be lost during the PCR cleanup.
Lane 1: 1kb DNA ladder
Lanes 2-4: PCR Squared reactions (contain 5X rxn buffer, dNTP, secondary PCR reaction, Forward and Reverse primers, Q5, and nanopure water)
Analysis: The PCR squared reactions which amplified the reaction is shown on the 1% agarose gel. The 100bp DNA ladder was not in the -20 degrees freezer, so instead the 1kb DNA ladder was used. However, the size of the gene came out to be around 741 nt. Many copies were made because a lot of the products will eventually be lost in PCR cleanup.
PCR Primer Overlap - Secondary PCR
Objective: The purpose of this experiment was to insert the forward and reverse primers into the primary PCR reaction to piece them together.
Lane 1: Empty
Lane 2: 100 bp DNA ladder
Lane 3: Secondary PCR reaction.
Analysis: Lane 3, according to the image, showed the secondary PCR reaction with a size of the gene was about 741 nt, which is similar to the size of the gene from the output file.
Week 3 & 4
Jessica - good work - include a ladder image - Dr. B 092713
PCR Primer Overlap - Primary PCR
Objective: The purpose of this experiment was to fill in the gaps between the oligos and piece together a full length DNA for the gene by conducting PCR.
Lane 1: Empty
Lane 2: 100 bp DNA ladder
Lane 3: Primary PCR reactions on 1% agarose gel.
Analysis: The Lane 3 shows the overlapping of the DNA with the nucleotides filling in the gaps in the oligo mix. The gel needs to run longer so the reaction comes down and appears as a more smeared image.
Ordered Forward and Reverse Primers of Gene:
PCR Primer Overlap - Oligo Mix
Objective: The purpose of this lab was to prepare an oligo mix in order to synthesize the gene of interest in future experiments.
Gene: HP Alpha CA
Analysis: The primers that were ordered were put together to create an oligo mix for future experimentation. In the later steps, PCR will be utilized to fill in the gaps between the oligos and piece together a full length of DNA for the gene.
PCR Primer Design Tails for pNIC-Bsa4 Cloning
Objective: The purpose of this lab was to design a forward and reverse primer from the gene (Alpha Carbonate Dehydratase) sequence obtained from the CDS and to synthesize compatible ends for LIC. After designing the primers, the gene would be inserted into the pNIC-Bsa4 as the accepting vector and cut with enzyme BsaI for further expression
0.4 uM Oligo concentration
50 mM Na+
0.3 mM dNTPs
0 mM Mg++
Forward Primer:
5’ TACTTCCAATCCATGAAAAAAACCTTCCTG 3’
30 bp
GC Content=36.7%
0mM Mg2+ Tm=58.1°C
1.5mM Mg2+ Tm=65.8ºC
2mM Mg2+ Tm=66.4ºC
4mM Mg2+ Tm=67.5ºC
6mM Mg2+ Tm=68.1ºC
Reverse Primer:
5’ TATCCACCTTTACTGTTAACGGGTCTTTGCAGA 3’
33 bp
GC Content=42.4%
0mM Mg2+ Tm=62.4°C
1.5mM Mg2+ Tm=69.8ºC
2mM Mg2+ Tm=70.3ºC
4mM Mg2+ Tm=71.3ºC
6mM Mg2+ Tm=71.8ºC
pNIC-Bsa4 Sequence:
Analysis: The coding sequence for Alpha Carbonate Dehydratase was used along with both upstream and downstream primers to perform LIC cloning. The sequence was then inserted in the pNIC-Bsa4 and was cut with enzyme BsaI, giving two restriction sites. The forward primer has a GC content of 36.7% with 30bp. The reverse primer had a GC content of 42.4% with 33bp and had a much higher melting point of 70.3 degrees Celcius than that of the forward primer.
Restriction Enzyme Digest
Objective: The purpose of this lab was to digest the pGBR22 plasmid with certain restriction enzymes - EcoRI, PvuII, and both, and to visualize the restriction sites on the agarose gel.
Lane 1: Empty.
Lane 2: 1kb DNA ladder.
Lane 3: Uncut plasmid containing diluted plasmid and nanopure water.
Lane 4: pGBR22 plasmid cut with EcoRI.
Lane 5: pGBR22 plasmid cut with PvuII.
Lane 6: pGBR22 plasmid cut with EcoRI + PvuII.
Analysis: Compared to the 1kb DNA ladder, there seemed to be no plasmid present for the uncut plasmid. Lane 4, plasmid cut with EcoRI, showed the plasmid at 4.0kb with one restriction site. Lane 5, cut with enzyme PvuII, showed the plasmid at 3.0kb and 1.0kb - two restriction enzymes. Lane 6, cut with EcoRI and PvuII, showed the plasmid at three restriction sites - 3.0kb, slightly below 1.0kb and slightly below 0.5kb. The intensity of the UV light appeared strongest where the DNA was most present.
PCR Trial 2
Lane 1:Empty.
Lane 2: 100bp DNA ladder.
Lane 3: Tube A consisting 1:1000 dilution template with 18 uL of nanopure water; 0.3ng of plasmid.
Lane 4: Tube B consisting 1:1000 dilution template with 9 uL of nanopure water; 3ng of plasmid.
Lane 5: Tube C consisting 1:100 dilution template with 9 uL of nanopure water; 30ng of plasmid.
Lane 6: Tube D consisting of no DNA template.
Analysis:
The size of the DNA is around 1000bp. The intensity of the UV light is stronger in lanes B and C because more DNA plasmid is present in those locations. The pGBR22 also consisted of M13 Forward and M13 Reverse primers.
Week 1 & 2
PCR Primer Design for Primer Overlap Assembly PCR
Objective:
The purpose of the experiment was to design a set of oligo primers of both reverse and forward for further experimentation.
Figure 1. Logfile of Helicobacter pylori ACD.
Protein length: 416
Total size of gene: 1248 nt
DNA Sequence:
1 ATGAAAAAAACCTTTCTGATCGCGCTCGTACTCGCGACTTCTCTCATCGGTGCGGAAAAC
61 GCGAAGTGGGATTACAAAAACAAAGAGAACGGTCCGCATCGTTGGGATAAGCTGCACAAA
121 GACTTCGAGGTATGCAAATCTGGTAAGTCTCAGTCTCCGATCAACATCGAGCACTATTAC
181 CATACCCAGGACAAAGCGGATCTGCAATTTAAGTACGCGGCGTCTAAACCTAAAGCAGTT
241 TTCTTCACCCACCACACCCTCAAAGCATCTTTCGAACCGACCAACCACATCAATTACCGT
301 GGTCACGACTACGTTCTGGACAACGTTCACTTCCACGCGCCGATGGAGTTCCTCATCAAC
361 AACAAGACCCGTCCTCTCAGCGCGCACTTCGTTCACAAGGACGCGAAAGGTCGTCTGCTG
421 GTACTGGCGATCGGTTTCGAAGAAGGTAAAGAAAATCCGAACCTCGATCCGATCCTGGAA
481 GGTATCCAGAAAAAACAGAACTTCAAAGAAGTTGCGCTCGACGCGTTCCTGCCGAAATCC
541 ATCAACTACTACCACTTCAACGGTTCTCTGACTGCCCCTCCGTGCACCGAAGGTGTTGCG
601 TGGTTTGTTATCGAGGAACCGCTGGAAGTTTCCGCCAAGCAGCTGGCGGAGATCAAAAAA
661 CGTATGAAGAACTCTCCAAACCAGCGTCCGGTTCAGCCGGACTACAACACTGTTATCATC
721 AAATCTTCTGCGAAAACTCGTCTCGGCGACTCCCCAGGTTACGTTATGTCTAATATCGAA
781 TTCCGTCAACTCACTCGCGGTCACTCTCCGTCTGACGAACGTGAAGCGCGTCGTGTAGAA
841 GAGGCGGGTGGTCAGCTGTTCGTTATCGGTGGTGAACTCCGCGTCAACGGCGTTCTGAAC
901 CTGACCCGTGCTCTGGGCGACGTGCCGGGTCGTCCGATGATCTCTAATGAACCAGAAACC
961 TGTCAGGTGCCTATTGAATCTTCCGACTACCTGGTTCTGCTCGCTTGCGACGGCATTTCT
1021 GACGTGTTCAACGAACGCGACCTGTACCAACTGGTTGAAGCCTTTGCGAATGACTACCCG
1081 GTGGAAGACTACGCGGAACTGTCTCGTTTCATCTGCACGAAAGCGATTGAAGCGGGTAGC
1141 GCGGATAACGTTTCTGTCGTCATTGGTTTCCTGCGTCCGCCGCAAGACGTTTGGAAACTG
1201 ATGAAGCACGAATCCGACGACGAAGATAGCGACGTCACCGACGAGGAG
Oligotide Sequence:
1 ATGAAAAAAACCTTTCTGATCGCGCTCGTACTCGCGACTTCTCTCATCGGTGCGGAA 57
2 GATGCGGACCGTTCTCTTTGTTTTTGTAATCCCACTTCGCGTTTTCCGCACCGATGAGAGA 61
3 AAAGAGAACGGTCCGCATCGTTGGGATAAGCTGCACAAAGACTTCGAGGTATGCAAATCTG 61
4 GGTAATAGTGCTCGATGTTGATCGGAGACTGAGACTTACCAGATTTGCATACCTCGAAGTC 61
5 GATCAACATCGAGCACTATTACCATACCCAGGACAAAGCGGATCTGCAATTTAAGTACGCG 61
6 GGTGTGGTGGGTGAAGAAAACTGCTTTAGGTTTAGACGCCGCGTACTTAAATTGCAGATCC 61
7 TTTTCTTCACCCACCACACCCTCAAAGCATCTTTCGAACCGACCAACCACATCAATTACCG 61
8 CGCGTGGAAGTGAACGTTGTCCAGAACGTAGTCGTGACCACGGTAATTGATGTGGTTGGTC 61
9 ACGTTCACTTCCACGCGCCGATGGAGTTCCTCATCAACAACAAGACCCGTCCTCTCAGCGC 61
10 GCCAGTACCAGCAGACGACCTTTCGCGTCCTTGTGAACGAAGTGCGCGCTGAGAGGACGGG 61
11 TCGTCTGCTGGTACTGGCGATCGGTTTCGAAGAAGGTAAAGAAAATCCGAACCTCGATCCG 61
12 ACTTCTTTGAAGTTCTGTTTTTTCTGGATACCTTCCAGGATCGGATCGAGGTTCGGATTTT 61
13 AGAAAAAACAGAACTTCAAAGAAGTTGCGCTCGACGCGTTCCTGCCGAAATCCATCAACTA 61
14 GGTGCACGGAGGGGCAGTCAGAGAACCGTTGAAGTGGTAGTAGTTGATGGATTTCGGCAGG 61
15 GCCCCTCCGTGCACCGAAGGTGTTGCGTGGTTTGTTATCGAGGAACCGCTGGAAGTTTCCG 61
16 GGAGAGTTCTTCATACGTTTTTTGATCTCCGCCAGCTGCTTGGCGGAAACTTCCAGCGGTT 61
17 TCAAAAAACGTATGAAGAACTCTCCAAACCAGCGTCCGGTTCAGCCGGACTACAACACTGT 61
18 GGGGAGTCGCCGAGACGAGTTTTCGCAGAAGATTTGATGATAACAGTGTTGTAGTCCGGCT 61
19 GTCTCGGCGACTCCCCAGGTTACGTTATGTCTAATATCGAATTCCGTCAACTCACTCGCGG 61
20 CCTCTTCTACACGACGCGCTTCACGTTCGTCAGACGGAGAGTGACCGCGAGTGAGTTGACG 61
21 CGCGTCGTGTAGAAGAGGCGGGTGGTCAGCTGTTCGTTATCGGTGGTGAACTCCGCGTCAA 61
22 ACCCGGCACGTCGCCCAGAGCACGGGTCAGGTTCAGAACGCCGTTGACGCGGAGTTCACCA 61
23 GCGACGTGCCGGGTCGTCCGATGATCTCTAATGAACCAGAAACCTGTCAGGTGCCTATTGA 61
24 AAATGCCGTCGCAAGCGAGCAGAACCAGGTAGTCGGAAGATTCAATAGGCACCTGACAGGT 61
25 CGCTTGCGACGGCATTTCTGACGTGTTCAACGAACGCGACCTGTACCAACTGGTTGAAGCC 61
26 GACAGTTCCGCGTAGTCTTCCACCGGGTAGTCATTCGCAAAGGCTTCAACCAGTTGGTACA 61
27 GAAGACTACGCGGAACTGTCTCGTTTCATCTGCACGAAAGCGATTGAAGCGGGTAGCGCGG 61
28 ACGTCTTGCGGCGGACGCAGGAAACCAATGACGACAGAAACGTTATCCGCGCTACCCGCTT 61
29 GTCCGCCGCAAGACGTTTGGAAACTGATGAAGCACGAATCCGACGACGAAGATAGCGACGT 61
30 CTCCTCGTCGGTGACGTCGCTATCTTCGTCGT 32
Quantifying DNA Using Nanodrop
Objective:
The purpose of this experiment was to determine the concentration and absorbance of the DNA plasmid.
Results/Conclusion:
The average concentration of the protein pGBR22 came out to be 397.6 ng/uL, which is pretty close to the originally obtained concentration of 365 ng/uL. Also, the purity of the sample in relation to protein (260/280) came out to an average of 1.88 and the purity in relation to other contaminants (260/230) came out to an average of 2.50. Thus, the protein is relatively pure.
Submit to DNA Sequencing
pGBR22 Forward Primer Sequence:
NNNNNNNNNNNNNGGNGANTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGNTTTTAGTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTAGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTNGNAAANCTGTCNTGCCAGCTGCNTNATGAATCGGCNANGCGCGGGGANANGCNGNTNGCGTATTGGGCGCTCNTCNNTTCCTCNNTCANTGACTCNNNNCNCTNGNCNTNNNNNNNNNNNNNGNNTCANCTCNNTNNANGNGNANTANNNNNNNNNNNNNNCNNGNNNANNNNNNANNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAAANNNCNNNNNNNNNNNNNNTTTTNNNNNNNNNNNN
pGBR22 Reverse Primer Sequence
NNNNNNNNNNNNNNNGANNATAGAATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANANGCCCGCACCGATCGCCCNTNCCCANCAGTNNNNCAGCCTGNATGGCNANGGNNGCNNCCTGNANNGNGCATTANNNCGGNGGGNGNNGNNGNNCGCNNANCGNGACNNTANNNNNNNGCNCCNNANNNNNNNCNTNNGNTTNNNNNNNNTTNNCNNNNNNTNNNNNTTNCCNNNAGNNNTAANNNGGGNTNNNTNNNNNNNNATTNNNGNNNNNNNNNNACNNNNNNNNNNNGNNNNNNGGNNNNNNNNNNNNNNNNNNNNNNNNCCNNNN
PCR
Objective:
The purpose of this lab was to, by using the Forward and Reverse Primers, intensify the coding sequence of the purple protein, and also to test four different samples by diluting the DNA primer.
Figure1 . Agarose gel consisting the gel ladder and four different samples dilutions of DNA primer.
Lane 1: Empty
Lane 2: 100 bp ladder
Lane 3: Tube A consisting 1:1000 dilution template with 18 uL of nanopure water.
Lane 4: Tube B consisting 1:1000 dilution template with 9 uL of nanopure water.
Lane 5: Tube C consisting 1:100 dilution template with 9 uL of nanopure water.
Lane 6: Tube D consisting of no DNA template.
Analysis/Conclusion:
After running the gel, the agarose gel went through the UV imaging box. By comparing to the ladder, the diluted primers seem to be the size of 100bp. However, the DNA may not have been diluted correctly because there are so much DNA present on top.
Analyzing DNA Sequencing
Objective:
The purpose of this lab was to analyze and determine the DNA sequence of the plasmid by using BLAST.